Virulence analysis of successively passaged scuticociliate strains and preparation and evaluation of whole-parasite vaccines from revitalized strains.

Scuticociliatosis is a major disease in marine fish aquaculture. Previous studies have demonstrated that scuticociliate vaccines can significantly enhance fish immunity. During vaccine preparation, the virulence of in vitro cultured scuticociliates undergoes significant changes. This study focuses on Pseudocohnilembus persalinus, a prevalent scuticociliate strain in northern marine fish, employing DIA-based proteomic analysis to compare protein profile differences between continuously passaged and reinvigorated scuticociliate groups. Additionally, whole-cell vaccines prepared from reinvigorated scuticociliates were used to immunize fish. The results showed that a total of 163 differentially expressed proteins were identified between the two groups. Upregulated proteins were mainly involved in metabolic pathways, while downregulated proteins were mostly associated with ribosomal function and energy metabolism. These differential proteins were primarily enriched in the phagosome signaling pathway, amino acid biosynthesis pathway, and glycolysis/gluconeogenesis pathway. Challenge experiments confirmed that the vaccine effectively inhibited parasite colonization. At 24 h, 48 h, and 72 h post-infection, the wound parasite reduction rates reached 99.2%, 94%, and 83.1%, respectively, with corresponding protection rates of 85.8%, 71.5%, and 57.2%. The vaccine significantly induced humoral immune responses in fish and enhanced early resistance to scuticociliate infection. This study provided valuable references for subsequent scuticociliates vaccine development and infection mechanism research.